1Division of Basic Biomedical Sciences, The Sanford School of Medicine of the University of South Dakota, Vermillion, South Dakota, USA.

Abstract

Streptococcus pyogenes Rgg is a transcriptional regulator that interacts with the cofactor LacD.1 to control growth phase-dependent expression of genes, including speB, which encodes a secreted cysteine protease. LacD.1 is thought to interact with Rgg when glycolytic intermediates are abundant in a manner that prevents Rgg-mediated activation of speB expression via binding to the promoter region. When the intermediates diminish, LacD.1 dissociates from Rgg and binds to the speB promoter to activate expression. The purpose of this study was to determine if Rgg bound to chromatin during the exponential phase of growth and, if so, to identify the binding sites. Rgg bound to 62 chromosomal sites, as determined by chromatin immunoprecipitation coupled with DNA microarrays. Thirty-eight were within noncoding DNA, including sites upstream of the genes encoding the M protein (M49), serum opacity factor (SOF), fibronectin-binding protein (SfbX49), and a prophage-encoded superantigen, SpeH. Each of these sites contained a promoter that was regulated by Rgg, as determined with transcriptional fusion assays. Purified Rgg also bound to the promoter regions of emm49, sof, and sfbX49 in vitro. Results obtained with a lacD.1 mutant showed that both LacD.1 and Rgg were necessary for the repression of emm49, sof, sfbX49, and speH expression. Overall, the results indicated that the DNA binding specificity of Rgg is responsive to environmental changes in a LacD.1-dependent manner and that Rgg and LacD.1 directly control virulence gene expression in the exponential phase of growth.

Noncoding DNA was enriched more than coding DNA. The level of DNA enrichment for each Rgg binding site was plotted, and the means were compared by using the Student t test. The difference was significant (P < 0.0001).

Gel shift assays of selected Rgg targets. Radiolabeled noncoding DNA (0.1 to 2 ng) upstream of emm49, sof, sfbX49, and speH was incubated with 0, 6.5, 26, 39, or 52 pmol of purified Rgg. Cold nonspecific DNA refers to the addition of unlabeled groEL DNA, and cold specific DNA refers to the addition of unlabeled competing DNA. A 250-fold excess of specific and nonspecific DNA was used in the control reactions.

Repression of speH (A), sof (B), sfbX49 (C), and emm49 (D) was dependent on both Rgg and LacD.1. Firefly reporter fusion plasmids containing the DNA upstream of speH, sof, sfbX49, and emm49 were transformed into the wild type (wt), rgg mutant (rgg−), lacD.1 mutant (lacD.1−), and lacD.1 mutant complemented with the lacD.1 gene (lacD.1+). The strains were grown to the exponential phase of growth, and promoter activity was determined by measuring luciferase. The means and standard errors of the means from three independent experiments are shown.

Growth phase modulation of Rgg binding specificity. In the exponential phase of growth, elevated glycolytic flux promotes a LacD.1 conformation (black circles) that forms a complex with Rgg (gray circles). Together, the proteins bind to Pemm49, PsfbX49, Psof, and PspeH to repress transcription. In the post-exponential phase of growth, decreased glycolytic flux causes a conformational change in LacD.1 that results in dissociation from Rgg and a change in the binding specificity of Rgg.